US6369212B1 - Cytochrome P450 gene highly expressed in the incompatible interaction - Google Patents

Abstract

The present invention related to a cDNA clone, designated to PepCYP (pepper cytochrome P450 gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof.

Description

BACKGROUND OF THE INVENTION

The present invention related to a CDNA clone, designated to PepCYP (pepper cytochrome P450 gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof

Colletotrichum gloeosporioides (Penz.) is the casual agent of anthracnose diseases on fruit crops (Daykin 1984; Dodd et al. 1991; Prusky et al. 1991) such as pepper (Capsicum annuum L.) (Kim et al. 1986; Manandhar et al. 1995). The infection of C. gloeosporioides is achieved through conidium germination and formation of appressorium and infection hyphae which are necessary for subsequent cuticular penetration (Bailey et al. 1992). In the avocado-C. gloeospoioides interaction, conidium germination and appressorium formation were similar on both unripe-resistant and ripe-susceptible fruits (Prusky and Saka 1989; Prusky et al. 1991). In the pepper and C. capsici pathosystem, germination and appressorium formation were higher on the unripe-resistant pepper fruit than on the ripe-susceptible fruit (Adikaram et al. 1983). The germination of C. musae was similar on both ripe-susceptible and unripe-resistant banana fruits, but the appressorium formation was stimulated on the unripe fruit (Swinburne 1976). Taken together, in these pathosystems, although conidium germination and appressorium formation are a prerequisite to infect the host plant, they may not be important factors to determine resistant or susceptible interactions between the host fruit of pre- or post-ripening stages and the Colletotrichum fungus. In contrast, plant responses to fungal morphogenesis during fruit ripening may be more important to determine resistant or susceptible interactions.

In Colletotrichum fungi, the topography of the plant surface was thought to be a physical signal for inducing appressorium formation (Staples and Macko 1980). The surface wax of avocado fruit was reported to act as a chemical signal for appressorium formation in C. gloeosporioides (Podila et al. 1993). In addition, the genes expressed during the appressorium formation of C. gloeosporioides induced by the host surface wax were cloned (Hwang and Kollatukudy 1995). However, plant responses to the conidium germination and appressorium formation of C. gloeosporioides have not been studied.

In a previous study, we found that an isolate of C. gloeosporioides showed an incompatible interaction with the ripe-red pepper fruit and a compatible interaction with the unripe-mature-green fruit (Oh et al. 1998), even though ripe fruits are generally susceptible to pathogen infection (Prusky et al. 1991; Swinburn 1983). In this pathosystem, higher levels of appressorium and infection hypha formation, and longer infection hypha were observed on the unripe fruit than on the ripe fruit in the early fungal infection step (Kim et al. 1999). A typical sunken necrosis was detected only in the unripe fruit, but not in the ripe fruit at 5 days after inoculation. Based on these data of the fungal morphogenesis and symptom development, we studied the gene expression in the pepper-C. gloeosporioides interaction.

By using a mRNA differential display method, we isolated several cDNAs that are differentially induced in the ripe fruit, but not in the unripe fruit after fungal infection. In this study, we report the characterization of one of these cDNAs encoding cytochrome P450 protein (PepCYP). To our knowledge, PepCYP is the first cytochrome P450 gene that accumulates to high levels in an incompatible plant-fungus interaction. The expression of PepCYP gene was examined based on fungal behaviors in the initial infection process and on symptom development during the differential interactions. We found that the PepCYP gene is induced by wounding or exogenous jasmonic acid during ripening. A possible role of the PepCYP in pepper fruits against pathogen infection during ripening is proposed.

SUMMARY OF THE INVENTION

The present invention relates to a cDNA clone, designated to a pepper cytochrome P450 gene, PepCYP, the sequence of which is depicted in SEQ ID No. 1. The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. By using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein SEQ ID NO:2 homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in compatible interaction, and then remains elevated in the incompatible interaction. However, in the compatible interaction the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 h and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process. The PepCYP gene can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with these genes. In addition, the PepCYP gene of this invention can be also used to produce transgenic plants that exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematode, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Restriction enzyme map of a cytochrome P450 cDNA, PepCYP, from pepper (Capsicum annuum). Numbers indicate nucleotide positions, and the open bar indicates the coding region that is 4 to 1509 nucleotide positions. The 3′ region of PepCYP clone contains the nucleotide sequence of the cDNA fragment, pddICC6, amplified by differential display. This partial cDNA fragment was used for gene-specific RNA probe for in situ hybridization.

FIG. 2. Alignment of the deduced amino acid sequence from PepCYP cDNA (pepper) (Genbank AF122821) with other cytochrome P450 proteins from potato (CYPs.ch), soybean (CYP71D8), avocado (CYP71A1), catmint (CYP71A5), Arabidopsis (CYP71B6), and tobacco (hsr515). The upper line indicates the hydrophobic N-terminal membrane anchor region of PepCYP (amino acid residues 1 to 27). The conserved PFGXGXRXCXG (SEQ ID NO: 3) heme-binding domain in the C-terminal region of the polypeptide is indicated by dots.

FIG. 3A-3B. A, Expression and induction of PepCYP gene from various organs of pepper by C. gloeosporioides inoculation or wounding. RNAs were isolated from the application sites of ripe fruit (R), unripe fruit (U), leaf, stem, and root at 24 h after the treatments of fungal inoculation (FI) or wounding (W). In addition, RNAs of both ripe (R′) and unripe fruits (U′) at 48 h after wounding were isolated. Ten μl of 5×10⁵ conidium/ml of C. gloeosporioides was used for drop-inoculation on various pepper organs. Organs treated with 10μl sterile-water except fungal spores for 24 h were used as the control (C). B, Induction of PepCYP gene from both ripe and unripe fruits of pepper by exogenous abscisic acid (ABA) or jasmonic acid (JA) treatments. RNAs were isolated from the application sites of both ripe (R) and unripe fruits (U) drop-applied with 10 μl of ABA(1=4μM, 2=40 μM) and JA (3=4 μM, 4=40 μM)for 24 h.

FIG. 4. Differential induction of PepCYP gene from the pepper fruit by C.gloeosporioides inoculation. RNAs were isolated from both ripe (RF/FUNGUS—the incompatible interaction) and unripe fruits (UF/FUNGUS—the compatible interaction) after the fungal infection with time-course. Water inoculation without fungal spores on both ripe (RF/WATER) and unripe fruits (UF/WATER) was used as the control. A cDNA for the PR-2 gene was hybridized to the same blot. Numbers indicate in h after inoculation.

FIG. 5A-5F. In situ localization of PepCYP mRNA in pepper fruits at 24 h and 72 h after inoculation (HAI) with C. gloeosporioides. The fungus with infection hypha started to invade in the outer epidermal cells of the unripe fruit at 24 HAI (B), and subsequently colonized the epidermal cells at 72 HAI (C). However, the fungal invasion was rarely observed in the ripe fruit at 24 (E) and 72 HAIs (F). Transverse sections were hybridized with the DIG-labeled antisense RNA probe of pddICC6 as a gene-specific probe. In both uninoculated unripe (A) and ripe fruits (D) used for the controls, transcripts were not detected in the epidermal and cortical cell layers. Deep-blue precipitation of transcripts were detected only in the epidermal cell layers (ec) of both unripe (B) and ripe fruits (E) at 24 HAI, and of the ripe fruit (F) at 72 HAI, but not in the cortical cell layers (cc). Transcripts were not detected in the unripe fruit that was colonized by the fungus at 72 HAI (C). A bar in (F) represents 100 μm. a, appressorium; cc, cortical cell layers; ec, epidermal cell layers; fc, fungal colonization; ih, infection hypha, s; spore.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has identified a cDNA clone, designated to PepCYP, from the incompatible interaction between pepper and the pepper anthracnose fungus Colletotrichum gloeosporioides using mRNA differential display and cDNA library screening.

The 1781 bp full-length sequence of PepCYP gene (FIG. 1) contains one open reading frame of 1506 bp from the first translation start (ATG) at nucleotide position 4 to a translational stop (TGA) at position 1509 (Genbank AF122821). The nucleotide sequences of pICC6 encode a polypeptide of 502 amino acids with a calculated molecular mass of 56.8 kDa. A putative polyadenylation site was identified at 22 bp downstream of the stop codon. The amino acid sequence of this cDNA is highly homologous to the genes encoding cytochrome P450s found in plants. Therefore, the pICC6 clone was designated PepCYP for pepper cytochrome P450. The PepCYP protein contains a hydrophobic membrane anchor region in the N terminal region (amino acid residues 1 to 27) (Bozak et al. 1990) (FIG. 2). A heme-binding domain (residues 435 to 440), PFGXGXRXCXG, (SEQ ID NO: 3) is located in the C terminal region of the polypeptide (Frey et al. 1995).

Sequence identity showed that the highest level was 59% with a potato cytochrome P450 protein (CYPs.ch) from a Solanum chacoense line rich in glycoalkaloids (Hutvágner et al. 1997) (FIG. 2). Sequence identity was 52% and 48% with CYP71D8 and CYP71D9 from soybean treated with an elicitor, respectively (Schopfer and Ebel 1998). The identities with other CYP71 subfamilies were 46% with avocado CYP71A1 (Bozak et al. 1990), 41% with catmint CYP71A5 (Clark et al. 1997), and 40% with Arabidopsis CYP71B6 (Mizutani et al. 1998). The minimum identity of amino acid sequence required to assign a cytochrome P450 within the same family should be higher than 40% (Nebert et al. 1991). Thus, the pepper gene belongs to the CYP71 family. In the tobacco and phytopathogenic bacterium Pseudomonas solanacearum interaction, the first cytochrome P450 gene hsr515 of tobacco that was expressed during the hypersensitive reaction was isolated (Czernic et al. 1996). The hsr515 protein shared 36% identity with the PepCYP.

We examined whether the expression of PepCYP gene was fruit-specific by fungal infection or inducible by other treatments. RNA gel blot analysis was performed with total RNAs prepared from fruits, leaves, stems, and roots of the pepper plants at 24 h after fungal inoculation or wounding. The expression of PepCYP gene was observed only in fruits, but not in leaves, stems, and roots after treatments (FIG. 3A). Interestingly, the PepCYP mRNA was induced in both ripe and unripe fruits by fungal infection, but wounding caused the induction of this mRNA only in the ripe fruit.

We further examined whether the wound-inducible PepCYP expression is inducible by ABA or JA treatments. RNA gel blot analysis was performed with total RNAs prepared from the application sites of both ripe and unripe fruits drop-applied with ABA or JA for 24 h. PepCYP mRNA highly accumulated only in the ripe fruit treated with JA at 40 μM (FIG. 3B). However, ABA did not affect the expression of PepCYP in both ripe and unripe fruits. To test whether a high concentration of JA is able to induce the expression of PepCYP in the unripe fruit, JA was applied to the unripe fruit at 100, 400, and 1000 μM. No induction of PepCYP expression was observed in the unripe fruit treated with JA (data not shown).

We examined whether the induction of time-course of PepCYP mRNA by C. gloeosporioides inoculation correlated with fungal morphogenesis and symptom development. RNA gel blot analysis was performed with both unripe and ripe fruits at 0, 3, 6, 12, 24, 48, and 72 HAIs. The PepCYP mRNA was not detected in both ripe and unripe fruits with water inoculation without fungal spores as a control. However, the accumulation of PepCYP mRNA was detected in both ripe and unripe fruits from 12 HAI (FIG. 4). In the unripe fruit, the expression of PepCYP gene is transient and peaks at 24 HAI before rapidly declining to barely detectable levels at 48 and 72 HAI. In contrast, in the ripe fruit, the expression level remains elevated. Thus, the results show that the PepCYP gene is inducible by fungal infection and is differentially expressed in compatible and incompatible interactions.

To examine the localization and accumulation of PepCYP mRNA during early infection, we performed in situ hybridization using a gene-specific antisense or sense RNA probe of pddICC6 (FIG. 1) with sections. The transverse-sections were prepared from the infection sites of both ripe and unripe fruits at 24 and 72 HAIs, respectively. The transcript of PepCYP was not detectable in uninoculated unripe (FIG. 5A) and ripe fruits (FIG. 5D) hybridized with anti-sense or sense RNA probe (data not shown). In unripe fruit, fungus with infection hypha started to invade outer epidermal cells at 24 HAI (FIG. 5B) (Oh et al. 1998). The accumulation of PepCYP mRNA at 24 HAI was localized only in the epidermal cells that were highly vacuolated, but not in the cortical cell layers (FIG. 5B). When the fungus colonized the outer epidermal cells at 72 HAL the induction-level of transcripts was very low or undetectable (FIG. 5C). In ripe fruit, fungal invasion was rarely observed at 24 HAI (FIG. 5E), and even at 72 HAI (FIG. 5F). This result shows that fungal invasion and colonization are inhibited in incompatible-ripe fruit during early infection. The accumulation of the transcripts in the epidermal cells at 24 HAI was sustained up to 72 HAI. These results suggest that the expression of the PepCYP gene is localized to the epidermal cell layers of the ripe fruit during incompatible interaction.

The PepCYP gene can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with these genes. In addition, the PepCYP gene of this invention can be also used to produce transgenic plants that exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematode, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.

EXAMPLES

Fungal inoculum and plant material

Monoconidial isolate KG13 of C. gloeosporioides was cultured on potato dextrose agar (Difco, Detroit, Mich.) for 5 days in darkness at 28° C. Sterile distilled water was added and conidia were harvested through four layers of cheesecloth to remove mycelial debris. Ten μl of 5×10⁵ conidium/ml of C. gloeospioides was used for drop-inoculation on both ripe and unripe pepper fruits as described (Oh et al. 1998).

Both ripe-red and unripe-mature-green fruits of pepper cv Nokkwang were grown and harvested under greenhouse conditions. For wound treatments, five healthy ripe and unripe fruits were deeply scratched with a knife and incubated at 100% relative humidity at 27° C. in the dark. Ten μl of ABA at 4 and 40 μM, or JA at 4 and 40 μM were drop-applied to both ripe and unripe sets of five fruits, respectively. After incubation under the conditions described above, the fruits were excised to 1 cm² at the drop-application site for the fungus, ABA or JA, and at the wounding site. The samples were then frozen in liquid nitrogen. Leaf, root, and stem samples were harvested from 3-week-old plants and handled as described above for fungal inoculation and wounding.

mRNA differential display

Total RNA was extracted from healthy or infected ripe and unripe fruits using the RNeasy Plant kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. We used total RNA as template for the reverse transcriptase reaction and performed differential display with [α³³P]dATP instead of [α³⁵S]dATP (Liang and Pardee 1992). Anchored primers and random-arbitrary primers were purchased from Operon Technologies (Operon, Alameda, Calif.). PCR-amplified cDNA fragments were separated on denaturing 5% polyacrylamide gels in Tris-borate buffer. cDNAs were recovered from the get, amplified by PCR, and cloned into pGEM-T easy vector (Promega, Madison, Wis.) as described (Oh et al. 1995).

Construction and screening of cDNA library

Poly(A)⁺mRNA was purified from total RNA of unripe-green fruits at 24 and 48 h after inoculation with C. gloeosporioides using the Oligotex mRNA Kit (Qiagen). The cDNA library (2.5×10⁵ plaque-forming unit with a mean insert size of 1.2 kb) was constructed in the cloning vector λZAPII (Stratagene, Heidelburg, Germany) according to the manufacturer's instructions.

A partial cDNA, designated pddICC6, from the differential display analysis was used as a probe to screen the C. gloeosprioides-induced pepper cDNA library. After three rounds of plaque hybridization, positive plaques were purified. The pBluescript SK phagemid containing cDNAs was excised in vivo from the ZAP Express vector using the ExAssit helper phage.

DNA sequencing and homology search

cDNA sequencing was performed with an ALFexpress automated DNA sequencer (Amersham Pharmacia Biotech, Buckinghamshire, UK). Analysis of nucleotide and amino acid sequences was performed using the DNASIS sequence analysis software for Windows, version 2.1 (Hitachi, San Bruno, Calif.). The multiple sequence alignment was produced with the clustal W program. For a homology search, cDNA sequence was compared to the NCBI non-redundant databases using the BLAST electronic mail server (Altschul et al. 1997).

RNA blot and hybridization

Total RNA (10 μg/lane) from each plant tissue used in this study was separated on 1.2% denaturing agarose gels in the presence of formaldehyde. RNA gel-blotting, hybridization and washing were conducted as described by the manufacturer of the positively charged nylon membrane employed (Hybond N⁺; Amersham Pharmacia Biotech). Radiolabeled probes were prepared with [α-³²P]dCTP (Amersham Pharmacia Biotech) using a random primer-labeling kit (Boehringer, Mannheim, Germany).

Tissue preparation and in situ hybridization

Pepper fruits were fixed in 1% glutaraldehyde/2% paraformaldehyde in 100 mM sodium phosphate buffer pH 7.0, dehydrated in ethanol and embedded in paraffin. Tissues were transverse-sectioned at 10 μm in thickness and stained with DAPI (10 μg/ml) to examine the infection hypha of the fungus in pepper fruits (Russell et al. 1975).

pddICC6 was used to prepare gene-specific DIG-labeled antisense RNA probes using T7 RNA polymerase or sense RNA probes using sp6 RNA polymerase. Hybridization steps were performed according to the manufacture's recommendation (Boehringer, Mannheim, Germany).

Cloning of a fungal-inducible cytochrome P450 gene of pepper

To isolate genes differentially induced from the ripe fruit but not from the unripe fruit in response to the fungal infection, we used mRNA differential display (Liang and Pardee 1992). Differential display was performed with total RNAs prepared from both unripe and ripe fruits at 24 and 48 h after fungal inoculation. The cDNAs amplified from the ripe fruit were excised from the gel, re-amplified, and cloned. RNA gel blot analysis with these clones was performed to confirm their differential expression. A cDNA clone, named pddICC6 for the incompatible Capsicum annuum/Colletotrichum gloeosporioides interaction, hybridized to a transcript of 1.8 kb which accumulated to high levels in the incompatible interaction (data not shown).

In order to isolate the full-length cDNA clone, the insert of pddICC6 was used as a probe for plaque hybridization using a cDNA library prepared from mRNA extracted from the unripe fruit at 24 and 48 h after inoculation with the fungus. A clone containing the longest insert from cDNA library screening was designated pICC6, isolated and sequenced. The 3′ region of pICC6 clone contained the nucleotide sequence of pddICC6 as expected.

Sequence analysis and characterization of PepCYP cDNA The 1781 bp full-length sequence (FIG. 1) contains one open reading frame of 1506 bp from the first translation start (ATG) at nucleotide position 4 to a translational stop (TGA) at position 1509 (GenBank AF122821). The nucleotide sequences of pICC6 encode a polypeptide of 502 amino acids with a calculated molecular mass of 56.8 kDa. A putative polyadenylation site was identified at 22 bp downstream of the stop codon. The amino acid sequence of this cDNA is highly homologous to the genes encoding cytochrome P450s found in plants. Therefore, the pICC6 clone was designated PepCYP for pepper cytochrome P450. The PepCYP protein contains a hydrophobic membrane anchor region in the N terminal region (amino acid residues 1 to 27) (Bozak et al. 1990) (FIG. 2). A heme-binding domain (residues 435 to 440), PFGXGXRXCXG, is located in the C terminal region of the polypeptide (Frey et al. 1995).

Sequence identity showed that the highest level was 59% with a potato cytochrome P450 protein (CYPs.ch) from a Solanum chacoense line rich in glycoalkaloids (Hutvágner et al. 1997) (FIG. 2). Sequence identity was 52% and 48% with CYP71D8 and CYP71D9 from soybean treated with an elicitor, respectively (Schopfer and Ebel 1998). The identities with other CYP71 subfamilies were 46% with avocado CYP71A1 (Bozak et al. 1990), 41% with catmint CYP71A5 (Clark et al. 1997), and 40% with Arabidopsis CYP71B6 (Mizutani et al. 1998). The minimum identity of amino acid sequence required to assign a cytochrome P450 within the same family should be higher than 40% (Nebert et al. 1991). Thus, the pepper gene belongs to the CYP71 family. In the tobacco and phytopathogenic bacterium Pseudomonas solanacearum interaction, the first cytochrome P450 gene hsr515 of tobacco that was expressed during the hypersensitive reaction was isolated (Czernic et al. 1996). The hsr515 protein shared 36% identity with the PepCYP.

Fruit-specific induction of PepCYP gene by fungal inoculation: up-regulation during ripening, and upon wounding and jasmonic acid treatments

C. gloeosporioides causes anthracnose diseases on the fruit of various plant species (Daykin 1984; Dodd et al. 1991; Kim et al. 1986, Manandhar et al. 1995, Prusky et al. 1991). Thus, we examined whether the expression of PepCYP gene was fruit-specific by fungal infection or inducible by other treatments. RNA gel blot analysis was performed with total RNAs prepared from fruits, leaves, stems, and roots of the pepper plants at 24 h after fungal inoculation or wounding. The expression of PepCYP gene was observed only in fruits, but not in leaves, stems, and roots after treatments (FIG. 3A). Interestingly, the PepCYP mRNA was induced in both ripe and unripe fruits by fungal infection, but wounding caused the induction of this mRNA only in the ripe fruit.

Jasmonic acid (JA) is a plant hormone with roles in mechanical wounding responses (Creelman et al. 1992; Creelman and Mullet 1997). ABA is hypothesized to be a key component in wound-signaling cascade leading to the activation of a defense gene (Pena-Cortés et al. 1996; Wasternack and Partheir 1997). Thus, we further examined whether the wound-inducible PepCYP expression is inducible by ABA or JA treatments. RNA gel blot analysis was performed with total RNAs prepared from the application sites of both ripe and unripe fruits drop-applied with ABA or JA for 24 h. PepCYP mRNA highly accumulated only in the ripe fruit treated with JA at 40 μM (FIG. 3B). However, ABA did not affect the expression of PepCYP in both ripe and unripe fruits. To test whether a high concentration of JA is able to induce the expression of PepCYP in the unripe fruit, JA was applied to the unripe fruit at 100, 400, and 1000 μM No induction of PepCYP expression was observed in the unripe fruit treated with JA (data not shown).

Differential induction of PCYP gene in compatible and incompatible interactions

In our previous studies (Kim et al. 1999; Oh et al. 1998), higher levels of the appressorium and infection hypha formations were observed on the unripe fruit than on the ripe fruit at 12 h and 24 h after inoculation (HAI), respectively. Initial anthracnose symptoms were detected only on the unripe fruit after 48 HAL, and typical sunken necrosis occurred within 120 HAI. Thus, we examined whether the induction of time-course of PepCYP mRNA by C. gloeosporioides inoculation correlated with fungal morphogenesis and symptom development. RNA gel blot analysis was performed with both unripe and ripe fruits at 0, 3, 6, 12, 24, 48, and 72 HAIs. The PepCYP mRNA was not detected in both ripe and unripe fruits with water inoculation without fungal spores as a control. However, the accumulation of PepCYP mRNA was detected in both ripe and unripe fruits from 12 HAI (FIG. 4). In the unripe fruit, the expression of PepCYP gene is transient and peaks at 24 HAI before rapidly declining to barely detectable levels at 48 and 72 HAI. In contrast, in the ripe fruit, the expression level remains elevated. Thus, the results show that the PepCYP gene is inducible by fungal infection and is differentially expressed in compatible and incompatible interactions.

A cDNA for the PR-2 gene from Nicotiana glutinosa was hybridized to the same blots to serve as a molecular marker for the activation of plant defense responses. In the unripe fruit, a basal level of PR-2mRNA was not detected, but the accumulation of PR-2mRNA was detected at 12 HAI (FIG. 4). And a biphasic accumulation of PR-2 mRNA was observed at 12 and 72 HAIs. In contrast, in the ripe fruit, a basal level of PR-2 mRNA was detected. The expression of PR-2 gene was rapidly induced in the ripe fruit at 3 HA and reached a maximum at 48 HAI.

Localization of PepCYP mRNA during early infection

To examine the localization and accumulation of PepCYP mRNA during early infection, we performed in situ hybridization using a gene-specific antisense or sense RNA probe of pddICC6 (FIG. 1) with sections. The transverse-sections were prepared from the infection sites of both ripe and unripe fruits at 24 and 72 HAIs, respectively. The transcript of PepCYP was not detectable in uninoculated unripe (FIG. 5A) and ripe fruits (FIG. 5D) hybridized with anti-sense or sense RNA probe (data not shown). In unripe fruit, fungus with infection hypha started to invade outer epidermal cells at 24 HAI (FIG. 5B) (Oh et al. 1998). The accumulation of PepCYP mRNA at 24 HAI was localized only in the epidermal cells that were highly vacuolated, but not in the cortical cell layers (FIG. 5B). When the fungus colonized the outer epidermal cells at 72 HAI, the induction-level of transcripts was very low or undetectable (FIG. 5C). In ripe fruit, fungal invasion was rarely observed at 24 HAI (FIG. 5E), and even at 72 HAI (FIG. 5F). This result shows that fungal invasion and colonization are inhibited in incompatible-ripe fruit during early infection. The accumulation of the transcripts in the epidermal cells at 24 HAI was sustained up to 72 HAI. These results suggest that the expression of the PepCYP gene is localized to the epidermal cell layers of the ripe fruit during incompatible interaction.

Discussion

As a first step to investigate the molecular mechanisms involved in the incompatible interaction between the ripe fruit of pepper and C. gloeosporioides, several cDNAs were isolated that were differentially expressed in the ripe fruit by fungal infection, but not in the unripe fruit. In this study with one of these cDNAs, we showed the characterization of the PepCYP gene that encodes a protein homologous to plant cytochrome P450 (Bozak et al. 1990; Frey et al. 1995). Cytochrome P450s in plants are membrane-bound proteins involved in several metabolic pathways related to the defense mechanisms (Maule and Ride 1983; Kessmann et al. 1990). Some genes encoding these proteins are induced by wounding (Batard et al. 1997; Frank et al. 1996). In a plant-phytopathogenic bacterium interaction, the tobacco cytochrome P450 gene, hsr515, was isolated during hypersensitive reaction (Czernic et al. 1996). In this study of a fungal-plant interaction, a pepper cytochrome P450 gene, PepCYP, was differentially expressed in compatible and incompatible interactions. Transcript levels of the two interactions were very different with maintenance of elevated levels in the incompatible interaction and a very substantial reduction in the compatible interaction. Together with the hsr515gene in a bacterial-plant interaction, the isolation of PepCYP in the pepper and fungus interaction suggests a new role for cytochrome P450s in plant-pathogen interactions.

Sequence comparison showed that PepCYP protein shared highest homology to the CYPs.ch from a Solanum chacoense line rich in glycoalkaloids (Hutvágner et al. 1997) as well as CYP71D8 and CYP71D9 from soybean treated with an elicitor (Schopfer and Ebel 1998). A possible role of CYPs.ch was suggested to be involved in the synthesis of stress-inducible metabolites. CYP71D8 and CYP71D9 may have a variety of functional roles in terpenoid metabolism (Christoffersen et al. 1995). The antimicrobial sesquiterpenoid phytoalexin, capsidol (Chavez-Moctezuma and Lozoya-Gloria 1996; Watson and Brooks 1984), was synthesized in pepper challenged with fungus (Ward 1976) and an abiotic elicitor, UV light (Back et al. 1998). Therefore, these data raise the possibility that PepCYP functions in the pepper plant's defense against fungal infection. The expression of PepCYP gene in the pepper fruit in response to fungal inoculation and wounding (FIGS. 3 and 4) supports a possible role of PepCYP involved in the plant's defense mechanism.

The first cytochrome P450, CYP71A, in plants was identified during avocado fruit ripening (Bozak et al. 1990). In this study, a basal level of PepCYP mRNA was not detected in ripe or unripe fruits or other various organs of pepper. However, the induction of PepCYP was detected only in fruit after fungal inoculation (FIG. 3A). In addition, the expression of PepCYP was induced only in ripe fruit by wounding and JA treatment (FIG. 3A and B). Thus, these results suggest that PepCYP is developmentally and fruit-specifically regulated, and the induction is upregulated during fruit ripening in response to wounding and JA. JA is reported to have roles in mechanical wounding responses (Creelman et al. 1992; Creelman and Mullet 1997) and in activating genes for plant disease resistance (Johnson et al. 1989; Xu et al. 1994; Reinbothe et al. 1994). However, the role of JA during the fruit ripening has not been well studied, in contrast to ethylene (Theologis 1992). A few cases that methyl JA triggers the ripening process of climacteric fruits including tomato and apple with ethylene production were reported (Czapski and Saniewski 1992; Saniewski et al. 1987a, 1987b). However, the role of JA in nonclimacteric fruits such as pepper, grape and strawberry has not been reported.

Fruit ripening represents a genetically synchronized developmental process unique to plants. Generally, ripe fruit is accompanied by an increased susceptibility to pathogen infection (Prusky et al. 1991; Swinburn 1983). As one of the reproductive organs of the plants, the fruit must be protected from pathogens or abiotic stresses. PR proteins and several antifungal proteins that are responsible for the protection against pathogens during fruit ripening have been identified (Fils-Lycaon et al. 1996; Meyer et al. 1996; Salzman et al. 1998; Tattersall et al. 1997). In the present study, the expression of PepCYP gene was detected only in the ripe fruit after fungal inoculation or wounding. We propose that the PepCYP gene is involved in the defense mechanism for the ripe fruit in order to maintain fruit integrity and to protect seed maturation against biotic and abiotic stresses.

Initial and mature infection hypha of C. gloeosporioides developed on pepper fruits at 12 and 24 HAIs, respectively (Oh et al. 1998). PepCYP mRNA in the fruit started to accumulate from 12 HAI and increased at 24 HAI (FIG. 4). Thus, it is likely that PepCYP gene expression occurs when the fungus directly invades the fruit by infection hypha. In microscopic and in situ hybridization observations, although cells didn't directly contact with the fungus, the induction of PepCYP transcript was detected throughout the epidermal cell layers. This result suggests that transcripts are induced by plant-derived defense signals generated after the fungus invasion. On the other hand, the accumulation of PR-2 mRNA in the ripe fruit at 3 HAI when the fungus germinates suggests that this gene is induced early in the incompatible interaction by fungal elicitors rather than plant-derived signals. The induction of PepCYP and PR-2 mRNAs was observed to be higher and faster, respectively, in the incompatible interaction than in the compatible interaction. These similar phenomena have been reported for many other plant-pathogen interactions (Ebrahim-Nesbat et al. 1989; 1993). Thus, higher and faster expression of many defense genes including PepCYP and PR-2 may confer disease resistance for the ripe fruit against fungal infection.

In summary, the present study showed that active fungal invasion and colonization processes are suppressed in the incompatible-interacting ripe fruit. Notably, PepCYP mRNA accumulated to higher levels in the ripe fruit in response to the fungal infection. The transcript is mainly localized in the epidermal cell layers of the pepper fruit after the fungal inoculation. We suggest that the PepCYP gene product plays a critical role in the plant's defense mechanism against the fungal invasion and colonization of the epidermal cells of the fruit in the incompatible interaction. It remains to be elucidated how the cytochrome P450 protein provides an effective defense against the fungal infection in pepper.

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10 1 1781 DNA Capsicum annuum 1 tgaatggaga ttcaattcac caacttagtt gcgtttttgc tctttctctc cagcatcatt 60 cttctactca aaaaatggaa aacccaaaag ctaaacttac ctcctggtcc atggaaatta 120 ccttttattg gaagcctaca tcacttggca gtggcaggtc cacttcctca tcatggccta 180 aaaaatctag caaaacttta tgggccgctc atgcacttac gactcgggga aattcctacc 240 gtcatcattt cgtccccgcg aatggcgaag gaagtactaa aaactcacga cctcgctttc 300 gcaacgaggc cgaaacttgt ggtggctgac atcgtccatt atgatagtac ggatatagca 360 ttttctccat atggtgaata ctggaggcag attcgtaaaa tttgcatact cgaactcctt 420 agtgccaaga tggtcaaatt ctttagctca attcgccagg atgagctgtc gatgatggtc 480 tcatctatac gaaccatgcc aaattttccc gtcaacctta cagacaaaat attttggttt 540 acaagttcgg taacttgtag atcagctctg ggaaaaatat gtcgtgacca agacaaactg 600 ataattttca tgagggaaat aatatcattg acaggtggat ttagtattgc tgattttttc 660 cctacatgga aaatgctaca tgatgttggt ggttcaaaaa ctagactgct gaaggctcat 720 cgtaaaatcg atgagatttt ggaacatgta gtgaatgagc acaaacagaa tcgagcggat 780 ggccaaaagg gtaatggcga atttggcggt gaagatttga tcgatgtttt gctaagggtt 840 cgagaaagtg gagaagttca aatttccatc acggatgaca atatcaaatc aatattagtg 900 gacatgttct ccgctggatc tgaaacgtca tcgacaacta taatttgggc attagctgaa 960 atgatgaaga aaccaagtgt tctagcaaag gcacaagctg aagtgagaca agtcttgaag 1020 gaaaagaaag gttttcaaca aattgatctt gatgagttga agtacttgaa gttagtaatc 1080 aaagaaactc taaggatgca ccctccaatt cctctattag tccctagaga atgtatgaag 1140 gatacaaaga ttgacgggta caatatacct ttcaaaactc gagtcatagt taatgcatgg 1200 gcaattggac gagatcctga aagttgggat gaccctgaaa gcttttcccc agagagattc 1260 gagaatagtt ctgttgactt tcttggaagc catcatcaat ttattccatt tggtgcggga 1320 agaaggattt gtcctggaat gctttttggt ttagccaatg ttggacaacc attagctcaa 1380 ttactttatc acttcgatcg gaaactccct aatggacaaa gtcacgaaaa tttggacatg 1440 acggagtcac ctggaatttc tgcaacaaga aaggatgatc ttgttttgat tgccacccct 1500 tatgatcctt gaatgtattg agacagttgt agaaataaaa aagagggaga aaatagaaat 1560 ggatgctgct tccaggtcat ttttgttggg agaaatttca aacttcatca acgtaactat 1620 atatagtgtt tgctagagtt ggtttattta ccactctata tcgtatttgg tgtactcaat 1680 aaattgtttg gtgtattata ttacagataa tggattttca ttttcatgtt aaaaaaaaaa 1740 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1781 2 502 PRT Capsicum annuum 2 Met Glu Ile Gln Phe Thr Asn Leu Val Ala Phe Leu Leu Phe Leu Ser 1 5 10 15 Ser Ile Ile Leu Leu Leu Lys Lys Trp Lys Thr Gln Lys Leu Gln Leu 20 25 30 Pro Pro Gly Pro Trp Lys Leu Pro Phe Ile Gly Ser Leu Phe Phe Leu 35 40 45 Ala Val Ala Gly Pro Leu Pro His His Gly Leu Lys Gln Leu Ala Lys 50 55 60 Leu Tyr Gly Pro Leu Met His Leu Arg Leu Gly Glu Ile Pro Thr Val 65 70 75 80 Ile Ile Ser Ser Pro Arg Met Ala Lys Glu Val Leu Lys Thr His Asp 85 90 95 Leu Ala Phe Ala Thr Arg Phe Lys Leu Val Val Ala Asp Ile Val His 100 105 110 Tyr Asp Ser Thr Asp Ile Ala Phe Ser Pro Tyr Gly Glu Trp Thr Arg 115 120 125 Gln Ile Arg Lys Ile Cys Ile Leu Glu Leu Leu Ser Ala Lys Met Val 130 135 140 Lys Phe Phe Ser Ser Ile Arg Gln Asp Glu Leu Ser Met Met Val Ser 145 150 155 160 Ser Ile Arg Thr Met Pro Asn Phe Pro Val Asn Leu Thr Asp Lys Ile 165 170 175 Phe Trp Phe Thr Ser Ser Val Thr Cys Arg Ser Ala Leu Gly Lys Ile 180 185 190 Cys Arg Asp Gln Asp Lys Leu Ile Ile Phe Met Arg Glu Ile Ile Ser 195 200 205 Leu Thr Gly Gly Phe Ser Ile Ala Asp Phe Phe Pro Thr Trp Lys Met 210 215 220 Leu His Asp Val Gly Gly Ser Lys Thr Arg Leu Leu Lys Ala His Arg 225 230 235 240 Lys Ile Asp Glu Ile Leu Glu His Val Val Asn Glu His Lys Gln Asn 245 250 255 Arg Ala Asp Gly Gln Lys Gly Gln Gly Glu Phe Gly Gly Glu Asp Leu 260 265 270 Ile Asp Val Leu Leu Arg Val Arg Glu Ser Gly Glu Val Gln Ile Ser 275 280 285 Ile Thr Asp Asp Asn Ile Lys Ser Ile Leu Val Asp Met Phe Ser Ala 290 295 300 Gly Ser Glu Thr Ser Ser Thr Thr Ile Ile Trp Ala Leu Ala Glu Met 305 310 315 320 Met Lys Lys Pro Ser Val Leu Ala Lys Ala Gln Ala Glu Val Arg Gln 325 330 335 Val Leu Lys Glu Lys Lys Gly Phe Gln Gln Ile Asp Leu Asp Glu Leu 340 345 350 Lys Tyr Leu Lys Leu Val Ile Lys Glu Thr Leu Arg Met His Pro Pro 355 360 365 Ile Pro Leu Leu Val Pro Arg Glu Cys Met Lys Asp Thr Lys Ile Asp 370 375 380 Gly Tyr Asn Ile Pro Phe Lys Thr Arg Val Ile Val Asn Ala Trp Ala 385 390 395 400 Ile Gly Arg Asp Pro Glu Ser Trp Asp Asp Pro Glu Ser Phe Ser Pro 405 410 415 Glu Arg Phe Glu Asn Ser Ser Val Asp Phe Leu Gly Ser His His Gln 420 425 430 Phe Ile Pro Phe Gly Ala Gly Arg Arg Ile Cys Pro Gly Met Lys Phe 435 440 445 Gly Leu Ala Asn Val Gly Gln Pro Leu Ala Gln Leu Leu Tyr His Phe 450 455 460 Asp Arg Lys Leu Pro Asn Gly Arg Ser His Glu Asn Leu Asp Met Thr 465 470 475 480 Glu Ser Pro Gly Ile Ser Ala Thr Arg Lys Asp Asp Leu Val Leu Ile 485 490 495 Ala Thr Pro Tyr Asp Pro 500 3 11 PRT Artificial Sequence Description of Artificial Sequence Consensus heme-binding domain 3 Pro Phe Gly Xaa Gly Xaa Arg Xaa Cys Xaa Gly 1 5 10 4 502 PRT Capsicum annuum MOD_RES (1) variable or unknown amino acid 4 Xaa Glu Ile Gln Phe Thr Asn Leu Val Ala Phe Leu Leu Phe Leu Ser 1 5 10 15 Ser Ile Ile Leu Leu Leu Lys Lys Trp Lys Thr Gln Lys Leu Xaa Leu 20 25 30 Pro Pro Gly Pro Trp Lys Leu Pro Phe Ile Gly Ser Leu Phe His Leu 35 40 45 Ala Val Ala Gly Pro Leu Pro His His Cys Leu Lys Asn Leu Ala Lys 50 55 60 Leu Tyr Gly Pro Leu Met His Leu Arg Leu Gly Glu Ile Pro Thr Val 65 70 75 80 Ile Ile Ser Ser Pro Arg Met Ala Lys Glu Val Leu Lys Thr His Asp 85 90 95 Leu Ala Phe Ala Thr Arg Pro Lys Leu Val Val Ala Asp Ile Val His 100 105 110 Tyr Asp Ser Thr Asp Ile Ala Phe Ser Pro Thr Gly Glu Ile Trp Arg 115 120 125 Gln Ile Arg Lys Ile Cys Ile Leu Glu Leu Leu Ser Ala Lys Met Val 130 135 140 Leu Phe Phe Ser Ser Ile Arg Gln Asp Glu Leu Ser Met Met Val Ser 145 150 155 160 Ser Ile Arg Thr Met Pro Met Phe Pro Val Asn Leu Thr Asp Lys Ile 165 170 175 Phe Trp Phe Thr Ser Ser Val Thr Cys Arg Ser Ala Leu Gly Lys Ile 180 185 190 Cys Arg Asp Gln Asp Lys Leu Ile Ile Phe Met Arg Glu Ile Ile Ser 195 200 205 Leu Thr Gly Gly Phe Ser Ile Ala Asp Phe Phe Pro Thr Trp Lys Xaa 210 215 220 Leu Met Asp Val Gly Gly Ser Lys Thr Arg Leu Leu Lys Ala His Arg 225 230 235 240 Lys Ile Asp Glu Ile Leu Glu His Val Val Asn Glu His Lys Gln Asn 245 250 255 Arg Ala Asp Gly Gln Lys Gly Xaa Gly Glu Phe Gly Gly Glu Asp Leu 260 265 270 Ile Asp Val Leu Leu Arg Val Arg Glu Ser Gly Glu Val Gln Ile Ser 275 280 285 Ile Thr Asp Asp Asn Ile Lys Ser Ile Leu Val Asp Met Phe Ser Ala 290 295 300 Gly Ser Glu Thr Ser Ser Thr Thr Ile Ile Trp Ala Leu Ala Met Xaa 305 310 315 320 Met Lys Lys Pro Ser Val Leu Ala Lys Ala Gln Ala Glu Val Arg Gln 325 330 335 Val Leu Lys Glu Lys Lys Gly Phe Gln Gln Ile Asp Leu Asp Glu Leu 340 345 350 Lys Tyr Leu Lys Leu Val Ile Lys Glu Thr Leu Arg Met Met Pro Pro 355 360 365 Ile Pro Leu Leu Val Pro Arg Glu Cys Met Lys Asp Thr Lys Ile Asp 370 375 380 Gly Thr Asn Ile Pro Phe Lys Thr Arg Val Ile Val Asn Ala Asn Ala 385 390 395 400 Ile Gly Arg Asp Pro Glu Ser Trp Asp Asp Pro Glu Ser Phe Ser Pro 405 410 415 Glu Arg Phe Glu Asn Ser Ser Val Asp Phe Leu Gly Ser His His Gln 420 425 430 Phe Ile Pro Phe Gly Ala Gly Arg Arg Ile Cys Pro Gly Met Leu Phe 435 440 445 Gly Leu Ala Asn Val Gly Gln Pro Leu Ala Gln Leu Leu Tyr His Phe 450 455 460 Asp Pro Lys Leu Pro Xaa Gly Gln Ser His Glu Asn Leu Asp Met Thr 465 470 475 480 Glu Ser Pro Gly Ile Ser Ala Thr Arg Lys Asp Asp Leu Val Leu Ile 485 490 495 Ala Thr Pro Tyr Asp Pro 500 5 500 PRT Solanum tuberosum MOD_RES (24)..(25) variable or unknown amino acid 5 Met Gln Leu Val Ser Ile Phe Leu Phe Ile Ser Phe Leu Phe Leu Leu 1 5 10 15 Arg Lys Trp Lys Lys Tyr Leu Xaa Xaa Ser Gln Thr Lys Lys Leu Pro 20 25 30 Pro Gly Pro Trp Lys Leu Pro Phe Ile Gln Gly Met Phe His Leu Ala 35 40 45 Gly Gly Leu Pro His His Val Leu Arg Asp Leu Ala Glu Lys Tyr Gly 50 55 60 Pro Leu Met His Leu Gln Leu Gly Glu Val Ser Ala Val Val Val Thr 65 70 75 80 Ser Pro Met Met Ala Lys Gln Val Leu Lys Thr His Asp Ile Ala Phe 85 90 95 Ala Ser Arg Pro Lys Leu Leu Ala Met Asp Ile Ile Cys Tyr Xaa Arg 100 105 110 Arg Asp Ile Ala Phe Ser Pro Thr Gly Asp Ile Trp Arg Gln Met Arg 115 120 125 Lys Ile Cys Ile Met Glu Val Leu Ser Ala Lys Ser Val Arg Ser Phe 130 135 140 Ser Ser Ile Arg His Asp Glu Val Val Arg Leu Ile Asp Ser Ile Gln 145 150 155 160 Pro Cys Phe Thr Ser Gln Glu Leu Val Asn Phe Thr Glu Arg Ile Ile 165 170 175 Trp Phe Thr Ser Ser Met Thr Cys Arg Ser Ala Phe Gly Gln Val Leu 180 185 190 Lys Glu Gln Glu Val Phe Ile Lys Asp Ile Arg Glu Val Ile Ser Leu 195 200 205 Ala Glu Gly Phe Asp Val Ala Asp Ile Phe Pro Ser Tyr Lys Phe Leu 210 215 220 Met Gly Phe Gly Gly Ala Lys Gln Lys Leu Leu Xaa Ala His Arg Lys 225 230 235 240 Val Asp Ser Ile Val Glu Asp Val Ile Lys Glu His Lys Lys Asn Leu 245 250 255 Ala Thr Arg Lys Ser Asp Asp Ala Ile Gly Gly Glu Asp Leu Val Asp 260 265 270 Ala Leu Val Arg Leu Met Xaa Asp Lys Ser Leu Gln Phe Pro Ile Xaa 275 280 285 Asn Asp Asn Ile Lys Ala Val Ile Ile Asp Leu Phe Ala Ala Gly Thr 290 295 300 Glu Thr Ser Ser Thr Thr Thr Val Trp Ala Trp Ala Met Met Leu Lys 305 310 315 320 Lys Pro Ser Val Phe Ala Lys Ala Gln Ala Lys Val Arg Glu Ala Phe 325 330 335 Arg Asp Lys Val Thr Phe Asp Lys His Asp Val Glu Glu Leu Lys Tyr 340 345 350 Leu Lys Leu Val Ile Lys Glu Thr Met Arg Leu His Ala Pro Val Pro 355 360 365 Leu Leu Val Pro Arg Glu Cys Arg Glu Glu Thr Glu Ile Asn Gly Thr 370 375 380 Thr Ile Pro Val Lys Thr Lys Val Met Val Asn Val Asn Ala Leu Gly 385 390 395 400 Arg Asp Pro Lys Tyr Trp Asp Asp Ala Glu Ser Phe Lys Pro Glu Arg 405 410 415 Phe Glu Gln Cys Ser Ile Asp Phe Ile Gly Asn Asn Phe Glu Tyr Leu 420 425 430 Pro Phe Gly Gly Gly Arg Arg Ile Cys Pro Gly Ile Ser Phe Gly Leu 435 440 445 Ala Asn Val Tyr Leu Pro Leu Ala Gln Leu Leu Tyr His Phe Asp Trp 450 455 460 Lys Leu Pro Thr Gly Met Glu Pro Lys Asp Leu Asp Leu Thr Glu Ser 465 470 475 480 Ala Gly Ile Thr Ala Ala Arg Lys Gly Asp Leu Tyr Leu Ile Ala Thr 485 490 495 Pro His Gln Pro 500 6 504 PRT Glycine max MOD_RES (194) variable or unknown amino acid 6 Met Glu Tyr Ser Pro Leu Ser Ile Val Ile Thr Phe Phe Val Phe Leu 1 5 10 15 Leu Leu His Trp Leu Val Lys Thr Tyr Lys Gln Lys Ser Ser His Lys 20 25 30 Leu Pro Pro Gly Pro Trp Arg Leu Pro Ile Ile Gly Met Leu Phe Gln 35 40 45 Leu Ala Leu Ala Ala Ser Leu Pro Asp Gln Ala Ile Gln Lys Leu Val 50 55 60 Arg Lys Tyr Gln Pro Leu Met His Leu Gln Leu Gly Glu Ile Ser Thr 65 70 75 80 Leu Val Val Ser Ser Pro Lys Met Ala Met Glu Met Met Lys Thr His 85 90 95 Asp Val His Phe Val Gln Arg Pro Gln Leu Leu Ala Pro Gln Phe Met 100 105 110 Val Ile Gly Ala Thr Asp Ile Ala Phe Ala Pro Thr Gly Asp Ile Trp 115 120 125 Arg Gln Ile Arg Lys Ile Cys Ile Leu Glu Leu Leu Ser Ala Lys Arg 130 135 140 Val Gln Ser Phe Ser His Ile Arg Gln Asp Glu Asn Lys Lys Leu Ile 145 150 155 160 Gln Ser Ile His Ser Glu Ala Gln Ser Pro Ile Asp Leu Ser Gly Lys 165 170 175 Leu Phe Ser Leu Leu Gly Thr Thr Val Ser Arg Ala Ala Phe Gly Lys 180 185 190 Glu Xaa Asp Asp Gln Asp Glu Phe Met Ser Leu Val Arg Lys Ala Ile 195 200 205 Thr Met Thr Gly Gly Phe Glu Val Asp Asp Met Phe Pro Ser Leu Lys 210 215 220 Pro Leu His Leu Leu Thr Arg Gln Lys Ala Lys Val Glu Met Val Met 225 230 235 240 Gln Arg Ala Asp Lys Ile Leu Glu Asp Ile Leu Arg Lys His Met Glu 245 250 255 Lys Arg Thr Arg Val Lys Glu Gln Xaa Gly Ser Glu Ala Glu Gln Glu 260 265 270 Asp Leu Val Asp Val Leu Leu Xaa Leu Lys Glu Ser Gly Ser Leu Glu 275 280 285 Val Pro Met Thr Met Glu Asn Ile Xaa Ala Val Ile Trp Asn Ile Phe 290 295 300 Ala Ala Gly Thr Asp Thr Ser Ala Ser Thr Leu Phe Trp Ala Xaa Ser 305 310 315 320 Met Met Met Lys Met Pro Lys Val Lys Glu Lys Ala Gln Ala Glu Leu 325 330 335 Arg Gln Ile Phe Lys Gly Lys Glu Ile Ile Arg Lys Thr Asp Leu Glu 340 345 350 Glu Leu Ser Tyr Leu Lys Ser Val Ile Lys Glu Thr Leu Arg Leu Met 355 360 365 Pro Pro Ser Gln Leu Ile Pro Arg Glu Cys Ile Ile Ser Thr Asn Ile 370 375 380 Asp Gly Thr Glu Ile Pro Ile Lys Thr Lys Val Met Ile Asn Thr Trp 385 390 395 400 Ala Ile Gln Arg Asp Pro Gln Tyr Trp Ser Asp Ala Asp Pro Phe Ile 405 410 415 Pro Glu Arg Phe Asn Asp Ser Ser Ile Asp Phe Xaa Gln Asn Ser Thr 420 425 430 Glu Tyr Ile Pro Phe Gly Ala Gly Arg Arg Met Cys Pro Gly Met Thr 435 440 445 Phe Gly Leu Ala Ser Ile Thr Leu Pro Leu Ala Leu Leu Leu Tyr His 450 455 460 Phe Asn Trp Glu Leu Pro Asn Lys Met Lys Pro Xaa Asp Leu Asp Xaa 465 470 475 480 Asp Glu His Phe Gly Met Ile Val Ala Arg Lys Asn Lys Leu Phe Leu 485 490 495 Ile Pro Thr Val Tyr Glu Ala Ser 500 7 502 PRT Persea sp. MOD_RES (1) variable or unknown amino acid 7 Xaa Ala Ile Leu Val Ser Leu Leu Phe Leu Ala Ile Ala Leu Thr Phe 1 5 10 15 Phe Leu Leu Lys Leu Xaa Glu Lys Arg Glu Lys Lys Pro Xaa Leu Pro 20 25 30 Pro Ser Gly Pro Asn Leu Pro Asp Asp Gly Gly Leu Phe Gln Leu Gly 35 40 45 Xaa Leu Pro His His Ser Leu Arg Ser Leu Ala Met Glu Leu Gln Pro 50 55 60 Leu Ile Leu Asp His Leu Gln His Ile Pro Thr Leu Ile Val Ser Thr 65 70 75 80 Ala Lys Ile Ala Glu Glu Ile Leu Lys Thr His Asp Leu Ile Phe Ala 85 90 95 Ser Arg Pro Ser Thr Thr Ala Ala Arg Arg Ile Phe Ile Asp Cys Thr 100 105 110 Asp Val Ala Phe Ser Pro Thr Gly Glu Ile Trp Arg Gln Val Arg Lys 115 120 125 Ile Cys Val Leu Glu Leu Leu Ser Ile Lys Arg Val Asn Ser Tyr Arg 130 135 140 Ser Ile Arg Glu Glu Glu Val Gly Leu Met Met Glu Arg Ile Ser Gln 145 150 155 160 Ser Cys Glu Thr Gln Glu Ala Val Asn Leu Ser Ile Leu Leu Leu Leu 165 170 175 Ile Ser Ser Gly Thr Ile Thr Arg Val Ala Phe Gly Lys Lys Tyr Glu 180 185 190 Gly Glu Glu Glu Arg Lys Xaa Lys Phe Ala Asp Leu Ala Thr Glu Leu 195 200 205 Thr Thr Leu Met Gly Ala Phe Phe Val Gly Asp Tyr Phe Pro Ser Phe 210 215 220 Ala Xaa Val Asp Val Leu Thr Gly Met Asp Ala Arg Leu Lys Arg Xaa 225 230 235 240 His Gly Glu Leu Asp Ala Xaa Val Asp His Val Leu Asp Asp His Leu 245 250 255 Ile Ser Arg Lys Ala Xaa Gly Ser Asp Gly Val Glu Gln Lys Asp Leu 260 265 270 Val Asp Val Leu Leu Xaa Leu Gln Lys Asp Ser Ser Leu Gly Val His 275 280 285 Leu Asn Arg Asn Asn Leu Lys Ala Val Ile Leu Asp Met Phe Ser Gly 290 295 300 Gly Thr Asp Thr Thr Ser Val Thr Leu Val Trp Ala Leu Ala Met Asp 305 310 315 320 Ile Lys His Pro Asp Val Asn Glu Lys Ala Gln Gln Glu Val Arg Arg 325 330 335 Val Val Gln Lys Lys Ala Lys Val Ile Lys Glu Asp Leu His Gln Leu 340 345 350 His Tyr Leu Lys Leu Ile Ile Lys Glu Thr Leu Arg Asp Met Pro Val 355 360 365 Ala Pro Leu Leu Val Pro Arg Glu Ser Thr Arg Asp Val Val Ile Arg 370 375 380 Gly Thr His Ile Pro Ala Lys Thr Arg Val Phe Ile Asn Ala Asn Ala 385 390 395 400 Ile Gly Arg Asp Pro Lys Ser Trp Glu Asn Ala Glu Glu Phe Leu Pro 405 410 415 Glu Arg Phe Val Asn Asn Ser Val Asp Phe Leu Gly Gln Asp Phe Gln 420 425 430 Ile Ile Pro Arg Gly Ala Gly Arg Arg Gly Cys Pro Gly Ile Ala Phe 435 440 445 Gln Ile Ser Ser Val Glu Ile Ser Leu Ala Asn Leu Leu Tyr Trp Phe 450 455 460 Asn Trp Glu Leu Pro Gly Asp Leu Thr Lys Glu Asp Leu Asp Met Ser 465 470 475 480 Glu Ala Val Gly Ile Thr Val His Met Lys Phe Pro Leu Gln Leu Val 485 490 495 Ala Lys Pro His Leu Ser 500 8 509 PRT Nepeta mussinii MOD_RES (1) variable or unknown amino acid 8 Xaa Val Ser Leu Ser Tyr Phe Leu Ile Ala Leu Leu Asp Thr Leu Pro 1 5 10 15 Phe Leu Leu Phe Leu Asn Lys Trp Arg Arg Ser Tyr Ser Gly Lys Thr 20 25 30 Pro Pro Pro Ser Pro Pro Lys Leu Pro Val Ile Gly Gly Leu Phe Gln 35 40 45 Leu Gly Leu Tyr Pro His His Tyr Leu Gln Ser Leu Ser Arg Arg Tyr 50 55 60 Gly Pro Leu Met Gln Leu His Phe Gly Ser Val Pro Val Leu Val Ala 65 70 75 80 Ser Ser Pro Lys Ala Ala Arg Glu Ile Xaa Lys Asn Gln Asp Ile Val 85 90 95 Phe Ser Asn Arg Pro Lys Met Ser Ile Ala Asn Arg Leu Arg Arg Ala 100 105 110 Xaa Pro Asp Val Ala Phe Thr Gln Thr Gly Glu Ile Trp Arg Gln Ile 115 120 125 Arg Ser Ile Cys Val Leu Gln Leu Leu Ser Leu Lys Arg Val Gln Ser 130 135 140 Phe Arg Arg Val Arg Glu Glu Glu Thr Ser Ile Met Val Glu Lys Ile 145 150 155 160 Asn Gln Leu Gly Ser Glu Ser Ser Thr Pro Val Asn Leu Ser Glu Leu 165 170 175 Leu Leu Ser Leu Thr Asn Asp Val Val Cys Arg Val Thr Leu Gly Lys 180 185 190 Lys Tyr Gly Gly Gly Xaa Gly Ser Glu Glu Val Asp Lys Leu Lys Glu 195 200 205 Met Leu Thr Glu Ile Gln Asn Leu Asn Gly Ile Ser Pro Val Trp Leu 210 215 220 Phe Ile Pro Trp Leu Xaa Trp Thr Arg Arg Phe Asp Gln Val Asp Gln 225 230 235 240 Arg Val Asp Arg Ile Val Lys Ala Phe Asp Gly Ile Leu Xaa Ser Val 245 250 255 Ile Gln Glu His Leu Glu Arg Asp Gly Asp Lys Asp Asp Asp Gly Asp 260 265 270 Gly Ala Leu Asp Phe Val Asp Ile Leu Leu Gln Phe Gln Arg Glu Xaa 275 280 285 Lys Xaa Arg Ser Pro Val Glu Asp Asp Thr Val Lys Ala Leu Ile Leu 290 295 300 Asp Met Phe Val Ala Gly Thr Asp Thr Thr Ala Thr Ala Leu Xaa Xaa 305 310 315 320 Ala Val Ala Met Leu Ile Lys Lys Pro Arg Ala Met Lys Arg Leu Gln 325 330 335 Asn Glu Val Arg Glu Val Ala Gln Ser Lys Ala Glu Ile Glu Lys Glu 340 345 350 Asp Leu Glu Lys Met Pro Tyr Leu Lys Ala Ser Ile Lys Glu Ser Leu 355 360 365 Arg Leu His Val Pro Val Val Leu Leu Val Pro Arg Glu Ser Thr Arg 370 375 380 Asp Thr Asn Val Leu Gly Thr Asp Ile Ala Ser Gly Thr Arg Val Leu 385 390 395 400 Ile Asn Ala Asn Ala Ile Ala Arg Asp Pro Ser Val Trp Glu Asn Pro 405 410 415 Glu Glu Phe Leu Pro Glu Arg Phe Leu Asp Ser Ser Asp Asp Tyr Ile 420 425 430 Gln Leu His Ile Gly Leu Leu Pro Phe Gly Ala Gly Arg Arg Gly Cys 435 440 445 Pro Gly Ala Thr Phe Ala Val Ala Ile Asp Glu Leu Ala Leu Ala Lys 450 455 460 Asp Val Arg Xaa Phe Asp Phe Gly Leu Pro Asn Gly Ala Arg Met Glu 465 470 475 480 Glu Leu Asp Met Ser Glu Thr Ser Gly Met Thr Val His Lys Xaa Ser 485 490 495 Pro Leu Leu Leu Leu Pro Ile Pro His His Ala Ala Pro 500 505 9 504 PRT Arabidopsis sp. MOD_RES (1) variable or unknown amino acid 9 Xaa Val Ser Leu Leu Ser Phe Phe Leu Leu Leu Leu Val Pro Ile Phe 1 5 10 15 Phe Leu Leu Ile Phe Thr Lys Lys Ile Lys Glu Ser Lys Gln Asn Leu 20 25 30 Pro Pro Gln Pro Ala Leu Leu Pro Ile Ile Gly Asn Leu Phe Gln Leu 35 40 45 Gln Gly Leu Leu His His Cys Leu His Asp Leu Ser Lys Xaa His Gly 50 55 60 Pro Val Met His Leu Arg Leu Gly Phe Ala Pro Met Val Val Ile Ser 65 70 75 80 Ser Ser Ser Ala Ala Arg Glu Ile Met Lys Asn Gln Asp Ile Val Phe 85 90 95 Ser Asn Arg Pro Lys Met Ser Ile Ala Asn Phe Leu Phe Phe Gly Gly 100 105 110 Lys Asp Ile Gly Phe Gly Val Thr Gly Asp Glu Trp Arg Glu Leu Arg 115 120 125 Lys Leu Ser Val Arg Glu Phe Phe Ser Val Lys Ile Val Gln Ser Phe 130 135 140 Ile Tyr Ile Arg Glu Glu Glu Asn Asp Leu Met Ile Lys Lys Leu Lys 145 150 155 160 Glu Leu Ala Glu Lys Gln Ser Pro Val Asp Leu Ser Lys Ile Leu Phe 165 170 175 Gly Leu Thr Ala Ser Ile Ile Phe Arg Thr Ala Phe Gly Gln Ser Phe 180 185 190 Phe Asp Asn Lys Met Val Asp Gln Glu Ser Ile Lys Glu Leu Met Phe 195 200 205 Glu Ser Leu Ser Asn Met Thr Phe Arg Phe Ser Asp Phe Phe Pro Thr 210 215 220 Ala Gly Leu Lys Trp Phe Ile Gly Phe Val Ser Gly Gln His Leu Arg 225 230 235 240 Leu Tyr Xaa Val Phe Xaa Arg Val Asp Thr Ile Phe Xaa His Ile Val 245 250 255 Asp Asp His Met Ser Lys Lys Ala Thr Gln Asp Arg Pro Asp Met Val 260 265 270 Asp Ala Ile Leu Asp Met Ile Asp Asn Glu Gln Gln Tyr Ala Ser Phe 275 280 285 Lys Leu Thr Val Asp His Leu Lys Gly Val Leu Ser Asn Ile Tyr His 290 295 300 Ala Gly Ile Asp Thr Ser Ala Ile Ile Leu Ile Trp Ala Leu Ala Met 305 310 315 320 Leu Val Arg Xaa Pro Arg Val His Lys Lys Ala Gln Asp Glu Ile Arg 325 330 335 Thr Cys Ile Gln Ile Lys Gln Glu Gly Arg Ile Met Lys Glu Asp Leu 340 345 350 Asp Lys Leu Gln Tyr Leu Lys Leu Val Val Lys Glu Thr Leu Arg Leu 355 360 365 Met Pro Ala Ala Pro Leu Leu Leu Pro Arg Glu Thr Met Ala Asp Ile 370 375 380 Lys Ile Gln Gly Thr Asp Ile Pro Gln Xaa Arg Ala Leu Leu Val Asn 385 390 395 400 Ala Asn Ser Ile Gly Arg Asp Pro Glu Ser Trp Lys Asn Pro Glu Glu 405 410 415 Phe Asn Pro Glu Arg Phe Ile Asp Cys Pro Val Asp Tyr Leu Gly His 420 425 430 Ser Cys Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg Ile Cys Pro Gly 435 440 445 Ile Ala Met Ala Ile Ala Thr Ile Glu Leu Gly Leu Leu Met Leu Leu 450 455 460 Tyr Phe Phe Asp Trp Asn Met Pro Glu Lys Lys Lys Asp Met Asp Met 465 470 475 480 Glu Glu Ala Gly Asp Leu Ile Val Asp Lys Lys Tyr Pro Leu Glu Leu 485 490 495 Leu Pro Val Ile Arg Ile Ser Leu 500 10 509 PRT Nicotiana sp. MOD_RES (32) variable or unknown amino acid 10 Met Glu Gly Thr Asn Leu Thr Thr Tyr Ala Ala Val Phe Leu Gly Thr 1 5 10 15 Leu Phe Leu Leu Phe Leu Ser Lys Leu Leu Arg Gln Arg Lys Leu Xaa 20 25 30 Leu Pro Pro Gln Pro Lys Pro Trp Pro Ile Ile Gly Gly Leu Asn Leu 35 40 45 Ile Gly Asn Leu Pro His His Ser Ile His Glu Leu Ser Leu Leu Thr 50 55 60 Gly Pro Val Met Gln Leu Gln Phe Gly Ser Phe Pro Val Val Val Gly 65 70 75 80 Ser Ser Val Lys Met Ala Ile Ile Phe Leu Thr Ser Met Asp Ile Asn 85 90 95 Phe Val Gly Arg Pro Lys Thr Ala Ala Gly Xaa Tyr Thr Thr Ile Asp 100 105 110 Tyr Ser Asp Ile Thr Trp Ser Pro Thr Gly Pro Ile Trp Arg Gln Ala 115 120 125 Arg Lys Xaa Cys Leu Thr Glu Leu Phe Ser Thr Lys Cys Leu Asp Ser 130 135 140 Tyr Glu Tyr Phe Arg Ala Glu Glu Leu Asn Ser Leu Leu His Asn Leu 145 150 155 160 Met Lys Ile Ser Gln Lys Pro Ile Val Leu Lys Asp Tyr Leu Thr Thr 165 170 175 Leu Ser Leu Xaa Val Ile Ser Arg Met Val Leu Gly Lys Arg Tyr Leu 180 185 190 Asp Glu Ser Glu Xaa Ser Phe Val Xaa Pro Glu Glu Phe Lys Lys Met 195 200 205 Leu Asp Glu Leu Phe Leu Leu Xaa Gly Val Leu Xaa Ile Gly Asp Ser 210 215 220 Ile Pro Trp Ile Asp Phe Met Asp Leu Gln Gly Tyr Val Ile Arg Met 225 230 235 240 Lys Val Val Ser Lys Lys Phe Asp Lys Ile Leu Glu His Val Ile Asp 245 250 255 Glu His Xaa Ile Arg Arg Asn Gly Val Glu Xaa Tyr Val Ala Xaa Asp 260 265 270 Met Glu Asp Val Leu Leu Gln Ile Ala Asp Asp Pro Xaa Leu Glu Val 275 280 285 Lys Leu Glu Arg Glu Gly Val Lys Ala Phe Thr Gln Asp Met Leu Ala 290 295 300 Gly Gly Ile Glu Ser Ser Ala Val Thr Val Ile Trp Ala Ile Ser Met 305 310 315 320 Leu Ser Lys Lys Pro Glu Ile Phe Lys Lys Ala Thr Glu Glu Leu Asp 325 330 335 Arg Val Ile Gln Gln Asn Arg Trp Val Gln Lys Lys Asp Ile Pro Lys 340 345 350 Leu Pro Tyr Ile Glu Ala Ile Val Lys Glu Thr Met Arg Leu His Pro 355 360 365 Phe Ala Pro Met Lys Ala Thr Glu Cys Arg Glu Asn Ser Lys Val Ala 370 375 380 Gly Thr Asp Val Gln Lys Gly Thr Arg Val Leu Val Ser Val Asn Thr 385 390 395 400 Ile Gly Arg Asp Pro Thr Leu Trp Asp Glu Pro Glu Val Phe Lys Pro 405 410 415 Glu Arg Phe His Glu Lys Ala Ser Ile Asp Val Leu Gly His Glu Tyr 420 425 430 Gln Leu Leu Pro Pro Gly Val Gly Arg Arg Met Cys Pro Gly Tyr Ser 435 440 445 Leu Gln Leu Lys Val Ile Gln Ala Ser Leu Ala Xaa Leu Leu His Gly 450 455 460 Phe Asn Trp Ser Leu Pro Asp Asn Met Thr Pro Glu Asp Leu Asn Met 465 470 475 480 Asp Glu Ile Phe Gly Leu Ser Thr Pro Lys Lys Phe Pro Leu Ala Thr 485 490 495 Val Ile Glu Pro Pro Leu Ser Pro Lys Leu Thr Ser Thr 500 505

Claims (2)

What is claimed is:

1. An isolated nucleic acid molecule encoding the pepper cytochrome P450 protein (PepCYP) having the amino acid sequence of SEQ ID NO: 2.

2. The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule is a cDNA molecule having the nucleotide sequence of SEQ ID NO: 1.

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